BE1020835A5 - METHOD FOR MANUFACTURING A PLANT EXTRACT, I.H.B. FROM DESMODIUM. - Google Patents

Description

Method for the manufacture of a plant extract, i.h.b. from Desmodium

The present invention relates to a method for manufacturing a plant extract, in particular an extract from Desmodium, more in particular Desmodium adscendens, possibly of exotic origin.

Plants have been used for their medicinal properties since ancient times. Many present-day drugs are or are derived from active components of plants such as paclitaxel (Taxol) from Taxus brevifolia and morphine from Papaver somniferum. Many plants are also used in traditional medicine in Africa, including Desmódium adscendens. In terms of geographical distribution and use, the plant is native to many tropical or subtropical countries of Africa and South America, among others, and grows on open plains, meadows and along the road, so that this plant is freely accessible and therefore makes it attractive as a medicine. for populations where the drugs are often difficult to purchase due to their cost. Thus, in traditional medicine, the plant is used for, among other things, asthma, pain, fever, epilepsy, hepatitis and muscle cramps. For this, a decoct is used as a warm, aqueous extract of the leaves, branches or the stem.

There are a number of commercial preparations of flavonoid-containing leaf extracts from the plant in circulation, which are traded as food supplements with various properties that promote good health.

Medicinal plants have also been used for centuries to treat liver disorders. Thus, Silybum marianum, Picrorrhiza kurroa, Curcuma longa, Glycyrrhiza glabra, Phyllantus amarus and Andrographis paniculata are plants of which a hepatoprotective effect was suspected.

The liver is a very important organ in the human body that is located at the top right of the abdomen and can weigh 1.4 to 1.6 kg in adults. About 1.5 l of blood flows to the liver via the liver artery and the portal vein per minute, which corresponds to 28% of the heart rate. The drainage is done via the hepatic vein and bile ducts. The liver cells themselves are organized in irregular units, the lobes, with a central hepatic vein in the middle. At the ends of the lobules are the hepatic arteries and portal vein. The latter two form a kind of branching in the form of sinusoids, so that about 70% of the surface of the hepatocytes comes into contact with the blood vessels for maximum exchange. Approximately 15% of the hepatocyte membranes are in contact with the bile ducts, which also lie on the corners of the lobules. The bile produced by the liver is drained through bile ducts that open into the gallbladder.

The liver is responsible for metabolic homeostasis and plays a crucial role in carbohydrate and lipid metabolism. In addition, the liver is responsible for trans, resp. de-amination and synthesis of proteins such as transport proteins and clotting factors and storage of vitamins A, D and B12. Good bile secretion is important for resorption of fat-soluble vitamins.

Another important function is detoxification and excretion of endogenous waste, such as ammonia, and exogenous substances, such as pharmaceuticals.

Since the liver ensures many important tasks, liver disorders have a major influence on the body. Liver cirrhosis, for example, is one of the leading causes of death in the Western world. Furthermore, jaundice is a condition caused by an increase in unconjugated or indirect bilirubin or in conjugated or direct bilirubin, which gives rise to yellowing of the skin and sclerae. Causes of an increase in indirect bilirubin are increased bilirubin production such as heriditary spherocytosis, reduced uptake in the liver (competition with pharmaceuticals) and. impaired conjugation (Crigler-Najjar syndrome). Causes of increased direct bilirubin are a change in expression of membrane transporters of the canaliculi (Dubin-Johnson syndrome) and decreased excretion of bilirubin glucuronides (cholestasis).

Cholestase is a condition where conjugated bilirubin is drained away to the bile ducts and returns to the blood. Causes of intra-hepatic cholestasis are hepatocellular dysfunction - such as damage and swelling - in viral hepatitis, chronic hepatitis and liver cirrhosis. Medications such as estrogens can also be the cause of cholestasis.

Cirrhosis is a result of progressive necrosis with scarring (fibrosis), whereby nodular regeneration occurs between the scars and disrupts normal construction. Consequences are congestion, portal hypertension, encephalopathy, ascites, etc. 60 to 70% of cases of cirrhosis of the liver are caused by alcohol abuse. Other causes are viral hepatitis, biliary disorders and primary hemorchromatosis.

Liver failure is the final stage of massive necrosis of the liver due to viral hepatitis, drugs and chemicals, chronic liver diseases and hepatic dysfunction without necrosis, such as Reye's syndrome.

To situate the mechanisms of liver damage and to get an idea of cell damage, blood parameters are measured, such as the transaminases alanine aminotransferase (ALT) and aspartate aminotransferase (AST). ALT only occurs in the cytoplasm, while AST also occurs in the mitochondria. The transaminases rise rapidly because they are already released with cell wall damage. AST rises later than ALT and indicates that it is a more serious necrosé. Membrane enzymes such as gamma glutamyl transferase (yGT) and alkaline phosphates (AF) are found in the cell membranes of almost all body cells. Gamma-GT is increased in hepatitis of viral or bacterial origin, or in intoxication by drugs or alcohol. Alkaline phosphatase is also determined in the diagnosis and monitoring of liver disorders. These parameters give an idea of the liver damage.

The Desmodium adscendens plant referred to here includes flavonoids such as vitexin, rutin and isovitexin, the tetrahydroisoquinoline salsoline (3), soybean saponins including soybean saponin I (4), β-phenylethylamines such as tyramine (5) and hordenine shown below under (1) to ( 6) resp. and an indole-3-alkylamine.

Desmodium adscendens has several pharmacological properties. In the neuro-pharmacological field, an extract of the plant has a suppressing activity on the central nervous system. The ethanolic extract has analgesic and hypothermal activity and inhibits the propagation of tonic-clonic seizures.

Aqueous and ethanolic extracts from this plant reduce smooth muscle contractions and reduce the release of substances that activate smooth muscle cells into the lungs. Different fractions of the extract were investigated. A subtraction inhibits smooth muscle cell contraction induced by antigen, via inhibition of phospholipases which is achieved by activation of calcium activated potassium receptors. Saponins were present in this fraction. Furthermore, the fraction containing a tetrahydroisoquinoline analog inhibits the cytochrome P450 NADPH-dependent mono-oxygenase reaction, which produces epoxy and hydroxy eicosanoids. The fraction increases cox activity resulting in an increased prostaglandin production.

The European patent publication EP 0309342 describes the use of Desmodium in the treatment of hepatitis, and medicines for this. This patent describes the use of

Desmodium adscendens in the treatment of viral hepatitis of type A or B and toxic hepatitis, a priori not against all, i.h.b. for addicted people or for cancer treatment.

Until now, liver disease has been very difficult to treat and virtually impossible to prevent. This is also the problem that has arisen, how to prevent some liver disease.

The present invention has for its object to provide a solution to the aforementioned problem. M.a.w. the object of this invention is in the first instance the investigation of the hepatoprotective properties of extracts of Desmodium adscendens, in particular leaves, twigs and / or other above-ground but also underground parts thereof, and the preparation of a quantified extract therefrom.

When searching for a suitable substance or medicine for liver disorders, a substance from the group of inositols was tested. These form a class of compounds containing hexahydroxycyclohexane derivatives, in particular cyclic sugar alcohols.

Like sugars, inositols form part of the normal human diet, without being toxic. Several studies, including in humans, have shown that D-pinitol exerts an effect analogous to that of insulin to improve the required control of glycemia. The aforementioned studies do indeed suggest that there appears to be a synergy between D-pinitol and insulin at sub-maximal concentrations, but this is not obvious with glucose transport. It is understood that both isomers are meant here, including the non-active L-pinitol.

D-pinitol is a cyclic sugar alcohol with a low molecular weight. It is a methyl derivative of D-chiro-inositol. Both D-pinitol and D-chiro-inositol are structurally related to phosphatidyl inositols, which form a link in the insulin-induced signal transduction. D-pinitol, the structure of which is depicted below, occurs in many vegetables, soy products and pine trees - but not exclusively - and is metabolized in the body to chiro-inositol.

D-pinitol differs from the other inositols by its specific activity.

A further difference between D-pinitol and the other inositols is that it contains a methyl ether group. Given the aforementioned composition, D-pinitol has a natural influence on glucose metabolism, and hence on diabetes or diabetes. Indeed, in vitro pharmacological properties indicate efficacy of D-pinitol including hypoglycemic, antiatherogenic activity and the influence on the immune system. Regarding hypoglycemic activity, D-pinitol may improve glucose transport and insulin sensitivity. D.i. t.a. the fact that D-pinitol is partially metabolized in humans to D-chiro-inositol, which is actually the responsible substance for the biological activity: D-chiro-inositol has an effect on diabetes caused by partial metabolization from D-pinitol, the active compound for the positive effects, especially in type 2 diabetes. M.a.w. Insulin sensitivity or resistance is improved. Indeed, it is shown that D-pinitol has been metabolized in humans to D-chiro-inositol, i.h.b. with type 2 diabetes, and is also secreted unchanged.

In other words, it cannot be inferred at first sight that D-pinitol a priori cannot qualify as a useful, let alone a major element, to make a significant contribution to the prevention or treatment of the liver disorders referred to herein.

In contrast, there is the patent publication derived from EP 04723853 but nothing herein discloses the use of an extract from a plant containing D-pinitol to protect the liver function that points to some method of preventing, let alone treating, one or another liver disease; and certainly not an exotic plant using D-pinitol, or any extract that would contain this component.

Thus, according to the invention, a method has been proposed for producing pinitol from a plant extract, starting from a plant extract from a fraction of Desmodium adscendens, more i.h.b. leaves, branches and / or underground parts thereof, possibly also flowers or even fruits, which are dried beforehand.

Thanks to the invention, a standardized extract has thus been proposed derived from a known plant, Desmodium adscendens, quantified on the D-pinitol molecule in its action against hepatitis. The point is that the separation of D-pinitol from Desmodium adscendens referred to here is by no means known, while the Desmodium adscendens plant in question is used for numerous purposes. However, the framework of the protection sought can be placed back in the conscious and targeted choice of a specific indication thereof. Although there are indications in a neighboring domain of a whole range of products including flavonoids, amino acids and the like, there is no mention of D-pinitol, which is just responsible for the activity in Desmodium adscendens against liver disease.

Thus, thanks to the invention, an analytical method has been developed to obtain a preparation quantified for D-pinitol, in which biologically controlled isolation is proposed, in which the product is enriched in each case and numerous fractions are isolated and in which the resulting decoct additionally a large amount of D-pinitol. contains.

Due to the natural variation in the plants with regard to the constituents, it is necessary to guarantee the reproducibility and therefore to standardize the constituents.

The main components in Desmodium adscendens and the total amount of D-pinitol are determined by a suitable analytical method. The extraction process, the products obtained after extraction and the conditions are evaluated and optimized.

According to a preferred embodiment of the invention, the aforementioned Desmodium adscendens fraction is refined, i.h.b. in powder form.

According to a more preferred embodiment of the invention, an aqueous decoction of the aforementioned leaves and / or branches of Desmodium is then prepared to a decoct by boiling a certain amount of dried, optionally pulverized, leaves in a certain amount of water, i.h.b. distilled water, for a certain period of time, i.h.b. 5 x 200 g, 3 f and 1 hour respectively.

According to an even more preferred embodiment of the invention, the aforementioned aqueous decoction of leaves or branches of Desmodium is subsequently cooled, in particular after which the portions obtained are combined and filtered, after which the filtrate obtained is concentrated, i.h.b. under vacuum and then lyophilized or spray dried.

According to a particular embodiment of the invention, starting from 1 kg of dried leaves of 60 to 70 g, i.h.b. about 65 g of dry decoct.

According to an increasingly preferred embodiment of the invention, a certain amount, i.h.b. approx. 20 g, said decoct subjected to column chromatography, i.h.b. on Sephadex LH 2 O with methanol elution, with certain fractions, i.h.b. of 100 ml, collected and analyzed by thin layer chromatography, i.h.b. Silica gel, layer thickness 0.25 cm, MeOH / H2 O: 5: 1 as a mobile phase, and where their chromatographic pattern is determined.

According to a further embodiment of the invention, the aforementioned fractions are combined in a number of subfractions, i.h.b. 11, according to their chromatographic pattern.

According to a still further embodiment of the invention, a part selected for the aforementioned subfractions 5-11 [i.h.b. 200 mg] combined, i.h.b. those that exhibit a colored spot, this fraction being subjected to a further column chromatography, i.h.b. on Sephadex LH-20 eluted with MeOH, whereby again certain fractions, i.h.b. of 100 ml, collected and analyzed.

According to an ever further embodiment of the invention, the aforementioned subfractions 4-5, i.h.b. 120 mg combined from this column, subjecting them to a further column chromatography under the same conditions, after which a pure product is obtained, i.h.b. white.

According to a preferred embodiment of the invention, said extract is isolated and the isolated product is identified as the methylated cyclitol 3-O-methyl-chiro-inositol, also called (+) - D-pinitol or D-pinitol.

This invention also relates to a plant extract obtained according to a method as defined above for use in protecting the liver in a mammal, i.h.b. for the prevention of liver disease.

According to an additional embodiment of the invention, said plant extract is used for treatment of a liver disease in a mammal.

According to a private embodiment of the invention, the D-pinitol-containing plant extract is given to the mammal in the form of a composition containing it, said composition being selected from the group consisting of: a pharmaceutical composition, a food composition and / or a beverage composition .

Further characteristics and details of the method according to the invention are determined in the further sub-claims.

Further details are included in the following description of some preferred exemplary embodiments of the method according to the invention, which is explained in the light of the accompanying drawings.

Figure 1 represents a functional operating diagram of the method according to the invention in the form of a flow diagram.

Figure 2 is a realistic representation of a representative sample of leaves and flowers of Desmodium adscendens.

Figure 3 is a schematic representation of Insulin signal transduction.

Figure 4 is a graphical representation of the calibration line obtained after derivatization with BSTFA.

Figures 5 to 7 are each a graphical representation of concentration and surface ratios after 3 hours, respectively. derivatize after 6 hours and derivatize after overnight.

Figures 8 and 9 are each a graphical representation of the concentration and surface ratios after an hour of derivatization in a heat block.

Figures 10 and 11 are each a graphical representation of the D-pinitol / internal standard surface ratio per 100 mg sample for extraction under ultrasonic vibratory bath.

Figures 12 and 13 are each a graphical representation of the surface ratio of D-pinitol / internal standard per 100 mg of steel for extraction under a heated vibrating bath, respectively. extraction - reflux.

Figure 14 is a graphical representation of the average area ratios for the various extraction methods.

Figure 15 is a graphical representation of a response function.

Figure 16 is a graphical representation of malfunctions that shows an overview of residues. Figures 17 and 18 are each a graphical representation of individual measurements and averages on different days.

Figures 19 and 20 are each a graphical representation of the recovered values after application of the so-called standard addition method.

Figures 21 and 22 are each a partial chromatogram of the analysis without, respectively. with internal standard representing the voltage in mV i.f.v. minutes.

Figures 23 and 24 are each a full chromatogram of the analysis of the standards representing the voltage in mV i.f.v. minutes.

Figures 25 to 28 are each a mass spectrum for different substances or xylitol standards, respectively, representing the relative abundance in% i.f.v. the most intense signal in terms of specificity.

Figures 29 to 31 show Serum AST values 48h after acetaminophen administration; resp. Serum ALT values 48h and 72h after acetaminophen administration.

Figures 32 et seq. Show further experimental data.

This invention generally relates to a method, the various steps of which are described schematically and which are used for the production of an exotic plant extract, in particular Desmodium adscendens, of which the above and / or underground parts serve as a starting product in this production process.

Desmodium adscendens is an herb that belongs to the Fabaceae family and to the Desmodium genus, a fragment of which is shown in Figs. 2. It is a perennial that can reach a height of 0.5 to 1 m and has a round, hairy, creeping trunk with grooves. The plant is three-leafed. The supporting leaves are hairy to bare on the outside and 0.5-1 mm long and 1.5-3 mm wide. They are winter-proof. The petiole is hairy and 1 to 3 cm long.

The leaves are elliptical - inversely ovoid, blunt and bordered at the top and wedge-shaped - round at the base. The leaves are mainly bare on the top and thickly hairy on the bottom.

The inflorescence consists of axial and terminal clusters. The leaf spindle is grooved and abundantly hairy to fine hairy. The first bracts are tapered oval with a pointed top and 3.5 - 5 mm long and 1.5 - 2 mm wide. The flower stalks have the same hair as the leaf spindle and are 0.4 - 1.7 cm long. The petals are usually purple. The chalice has two lips and the chalice is finely hairy. The teeth of both lobes are hairy and 2 - 3 mm long. The crown flower is larger than the calyx and is oval.

The fruit has an extended flower stem of 0.5 - 2 mm long and is 1-5 empty, obliquely elongated with a size of 3.5 - 5.5 mm on 2.5 - 3 mm. The seed is transversely elliptical and 2.5 - 5 mm long and 1.5 mm wide.

In the experimental phase, a number of tests were first conducted in vitro, and then in vivo. The course of the test described below is shown schematically in the flow chart of Figure 1.

In a phytochemical study of Desmodium adscendens, plant material and its extraction from Ghana yielded an aqueous decoction of the leaves prepared by boiling 5 X 200 g of dried and pulverized leaves in 3 liters of distilled water for 1 hour. After cooling, the portions were combined and filtered. The filtrate was concentrated in vacuo and then lyophilized. Starting from 1 kg of dried leaves, typically about 65 g of dry decoct were obtained.

Then 20 g of decoct was subjected to column chromatography on Sephadex LH 2 O (120 X 4 cm) with methanol elution. 100 ml fractions were collected and analyzed by thin layer chromatography (silica gel Merck, layer thickness 0.25 cm, MeOH / H2 O: 5: 1 as a mobile phase). Spots were detected under UV light, in particular under a wavelength of 366 nm. After spraying with anisaldehyde 1% / H 2 SO 4 in MeOH, the plate was heated to 120 ° C for 10 minutes to obtain colored spots. Fractions were combined in 11 subfractions according to their chromatographic pattern. Subfractions 5-11 (200 mg) showed a spot with a green color, and were combined. This fraction was subjected to a further column chromatography on Sephadex LH 2 O (60 X 3 cm) eluted with MeOH, and again 100 ml fractions were collected and analyzed as described. Subfractions 4-5 (120 mg) of this column were combined, and after a new column chromatography under the same conditions, a crystalline product was obtained.

The phase of structural clarification by spectroscopic examination with 1 H, 13 C NMR and mass spectroscopy, and measurement of the specific optical rotation, led to the identification of the isolated product as the methylated cyclitol 3-O-methyl-chiro-inositol, also (+) - called pinitol or D-pinitol.

Treatment of 3T3-L1 adipocytes with 0.5 and 1 mM D-pinitol increases the mRNA expression of glucose transporter (GLUT4), insulin receptor substrate (1RS), peroxisome proliferator activated receptor Y (PPARγ) and CCAAT / enhancer-binding protein ( C / EBP). 1 mM D-pinitol increases adiponectin mRNA expression, an adipocytokine with anti-inflammatory, anti-diabetic and antiatherogenic properties, the expression of which is also increased by insulin. The increased expression of a number of factors can be explained by the insulin mimetic properties that D-pinitol possesses.

In L6 rat muscle cells, D-pinitol induces the translocation of GLUT4 to the cell membrane such as insulin and ensures the uptake of glucose (Fig. 3).

Regarding antiatherogenic activity, it was found that D-pinitol moderately inhibits foam cell formation by reduced secretion and expression of cytokines such as TNF-α, monocyte chemoattractant protein-1, IL-1beta, IL-8 and reduced expression of macrophage scavenger receptor, CD36 and CD86. The insulin mimetic activity of D-pinitol is probably also the explanation for this.

With regard to the influence on the immune system, it was established that D-pinitol has immunopharmacological properties and that D-pinitol reduces the expression of MHC-I, MHC-II, and costimulators such as CD80 and CD86, both in vitro and in vivo, by suppressing MAPKs activation and translocation of NF-kB and production reduces large amounts of IL-12 and proinflammatory cytokines in LPS-induced dendritic bone marrow cells. This results in the inhibition of the maturation of these cells. Treatment of dendritic cells with D-pinitol prevents these cells from inducing a normal cell mediated immune response and when LPS-stimulated dendritic cells are treated with D-pinitol, the proliferation of T cells and the production of INF-γ by CD4 + cells negatively affected. D-pinitol inhibits TNF-alpha expression in neutrophils. D-pinitol inhibits constitutive and induced NF-kB activation in a dose and time dependent manner. The inhibition is not cell specific and occurs through inhibition of IKK activation, IkBa degradation and phosphorylation, nuclear phosphorylation and translocation of p65. D-pinitol also reduces NF-kB dependent reporter gene expression and suppresses NF-kB dependent gene products involved in cell proliferation, anti-apoptosis, invasion and angiogenesis. This may explain why D-pinitol analogues, such as azole nucleoside analogues, have anti-tumor properties. Other derivatives of D-pinitol such as aminocyclitols inhibit glycosidase.

In vivo pharmacological properties were investigated in laboratory animals. In the in vivo tests performed, the liver was damaged, where it was investigated whether it works preventively and / or curatively. Until then, it has not yet been proven that the molecule in question has a curative effect, although its preventive nature has been proven. In addition to activity in diabetic mice resp. rats in streptozotocin-induced diabetic mice - where D-pinitol has an acute and chronic anti-hyperglycemic effect - increases basal uptake of 2-deoxyglucose in L6 muscle cells through intervention in insulin signal transduction. D-pinitol is not effective in severely insulin-resistant mice. In streptozotocin-induced diabetic rats, D-pinitol lowers blood glucose hemoglobin and increases insulin with D-pinitol also normalizing aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase liver values and having a lipid-lowering effect . The antioxidant effect is reflected in the reduction of lipid peroxidation and hydroperoxidation, an increase in non-enzymatic antioxidants and a normalization of the enzymatic antioxidants superoxide dismutase (SOD), glutathione peroxidase (GP), catalase and glutathione S-transferase ( GST).

Regarding the hepatoprotective effect, the substance normalizes aspartate .transaminase (AST), alanine transaminase (ALT) liver values and TNF-α values after induction of liver damage with galastosamine. In addition, D-pinitol reduces the. lipid peroxidation and normalizes the glutathione, glutathione reductase and glutathione peroxidase values.

There is also an anti-inflammatory effect, in which D-pinitol has anti-inflammatory properties in rat tests, both against acute (carrageenan-induced leg paw edema) and subacute (cotton pellet granuloma) inflammation. 'The substance also works antihelmintically.

Regarding the influence on the immune system demonstrated in. mice with OVA-induced asthma, the D-pinitol reduces the number of inflammatory cells in broncheoalveolar lavage fluid and reduces the infiltration of these cells in peric bronchiolar and perivascular regions. Inflammation in the lungs is therefore prevented. The Th2 cytokines such as IL-4, IL-5 and eotaxine decrease due to intake of D-pinitol and the Th1 cytokines such as INF-γ rise and so do the INF-γ positive CD4 cells. The Th1 / Th2 balance is further corrected by D-pinitol by increasing expression of the Th1 transcription factor T-bet and by decreasing the transcription factor GATA-3 which is increased in Th2 patologies. The gelatinolytic activity of MMP-9 in lung tissue , which is important in the migration of the inflammatory cells from blood to tissue, is reduced. D-pinitol thus reduces the hyperreactivity and inflammation seen in asthma.

Tests with people

In patients with type 2 diabetes, D-pinitol has a beneficial effect on fasting glucose values, HbA1c values and insulin. Total cholesterol, LDL / HDL ratio and systolic and diastolic blood pressure decrease after a 13-week treatment with twice 60 mg D-pinitol twice a day, while the HDL cholesterol levels rise. In patients with uncontrolled type 2 diabetes, a 12-week therapy with 20 mg / kg / day D-pinitol lowers, in addition to regular therapy, fasting and post-prandial glucose values as well as HbAc1 values, but does adiponectin; leptin, C-reactive protein (CRP) and free fatty acids do not change significantly. When D-pinitol is taken at a dose of 20 mg / kg / day over a shorter period of 4 weeks, the substance has no effect on basal and insulin-mediated glucose or lipid metabolism in insulin-resistant patients. In non-diabetic elderly patients, D-pinitol intake for 6 weeks has no effect on insulin-mediated glucose metabolism.

The development of an analytical method for D-pinitol in Desmodium adscendens decoct is shown below with the corresponding validation. Before a standardized preparation can be produced from plant material, the concentration of the active substance in the plant must be known. For this, a standard method is used that is repeatable, accurate, not time-consuming and also, preferably economically. The aim of this master's thesis is to develop an analytical method to determine the D-pinitol content in a decoct of Desmodium adscendens. Various parameters were investigated for this, such as the analysis technique (GC, HPLC, TLC), the column, the detector and any purification. When a possible method was developed, it would also be validated through the ICH standards. The linearity, range, repeatability, accuracy and specificity were investigated.

The materials used were methanol, HPLC grade with analytical grade, pyridine (99 +%) BSTFA 1% TMCS, standards D-pinitol (95%), xylitol (99% minimum) and dulcitol (99 +%), mannitol pa and sorbitol, helium, hydrogen gas and air, sodium chloride (pa), D - (+) - galactosamine hydrochloride (99% minimum) and sylimarin.

The decoct of Desmodium adscendens was prepared in the following way: 3 liters of distilled water were added to 200 grams of plant material. The whole was boiled for 1 hour after which it was cooled and filtered off. Volume reduction was achieved by evaporation using a rotavapor. As a final step, the decoct was lyophilized.

In the thin layer chromatography technique, the stationary phase is on a plastic or glass support. The stationary phase can be silica gel or modified silica, but also aluminum oxide, cellulose or kieselguhr.

1-10 μΙ of the solution to be analyzed was spotted about 1.5 cm from the bottom of the plate. The plate was then placed in a developing tank containing a layer of approximately 1 cm mobile phase. The mobile phase rose due to the capillary forces and, depending on the nature of the substances in the solution, the substances eluted faster or slower. When the liquid front almost reached the top of the plate, it was removed from the developing tank and the mobile phase evaporated. Afterwards a spot pattern could be made visible by UV light or by treatment with spray reagent and the retention times could be calculated. A qualitative determination can be made with the help of a densitometer that measures the intensity of the spots and converts them into a densitogram.

For the experiments, silica gel F254 kieselgel TLC plates, Lichrospher silica gel F254 HPTLC plates and silica gel 60 RP-18 F254 plates were used.

Gas chromatography GC is an analytical technique where separation between the stationary liquid phase and a mobile gas phase analytes. Due to the high temperature in the injection block, both the solvent and the analytes evaporate during the injection and condense on the colder column. By raising the column temperature, the relatively less volatile analytes also enter the gas phase and eventually reach the detector. The more non-polar and volatile the analytes, the more affinity for the gas phase and the shorter the retention time. More polar or less volatile analytes have more affinity for the more polar stationary phase and later reach the detector. Possible detectors are the flame ionization detector, electronic capture detector, nitrogen-phosphorus detector, catarometer, sulfur detector and mass spectrometer.

A GC-FIQ of the Tracé GC Ultra type with FID detector was used for the experiments. To verify specificity, a GC-MS of the Trace 2000 GC type was used with a voyager El MS detector.

A flame ionization detector (FID) and a mass spectrometer (MS) were used in these experiments. The flame ionization detector is a universal and sensitive detector. The eluate was mixed with H2 and air, and burned. As a result, organic compounds ionize and the ions increase the current strength relative to a constant-voltage collector electrode. The mass spectrometer works by ionizing molecules, after which the ions are measured.

Liquid chromatography is a further analytical technique in which the separation is by a difference in distribution between the stationary phase and the mobile liquid phase. When the stationary phase is more polar than the mobile phase, the term "normal phase chromatography" is used, while when the stationary phase is non-polar, the term "reversed phase chromatography" is used. The analytes were eluted with eluent compounds whose composition, and therefore also the polarity, can be changed to elute faster or slower analytes Possible detectors are UV detectors, Rl detectors, amperometric detectors, mass spectrometers and evaporative light scattering detectors (ELSD).

An ELSD detector was used for the experiments, where after the mobile phase evaporation, the light scattering was measured and converted into an electrical signal.

Possible methods of analysis of the analysis of sugars and sugar derivatives, including sugar alcohols such as cyclitols, are known. Several techniques are available for the determination of which a brief overview is given below.

For example GC techniques: because sugars and sugar derivatives are not volatile, a derivation step must also be added here. Both trimethylsilation and acetylation are known. As a detector, both a general flame ionization detector and a mass spectometer can be used.

Further HPLC techniques: since the analytes are not UV active, they must be derivatized before UV detection is possible. UV-active substances are made by, among other things, derivatization benzyl chloride or by means of. UV active ion pair reagents. In addition to UV detection, refractive index detection, pulsed amperometric detection, mass spectrometry or electron light scattering detection, without derivatization, can also be used. Both Ci8 columns and anion exchange columns can be used. When anion exchange columns are used, the sugars are converted to anions using a base.

Other possible techniques are enzymatic essays, capillary electrophoresis and thin layer chromatography densitometry. In thin layer chromatography described by Pothier, based on fingerprinting by this means, an attempt is also made to separate D-pinitol from the other substances in the decoct, because TLC methods are fast and relatively inexpensive.

D-pinitol (standard) and the sample were dissolved in distilled water and 50% methanol-50% water. As a mobile phase, ethyl acetate-formic acid-acetic acid-water (67.5: 7.5: 7.5: 17.5), chloroform - methanol - water (54.5: 36.5: 9), chloroform - methanol - water (55: 36: 9), chloroform - methanol - water (46.5: 46.5: 7) and chloroform - methanol - water (33: 53.5: 13.5).

D-pinitol was not visible under UV light on the plates with UV indicator F254S, not even at very high concentration (10 mg / ml), while using anisaldehyde or thymol as a spray reagent, multiple spots were seen with the standard.

As a result, it was decided to develop a method that uses a different analysis technique. Because sufficient GC methods are described in the literature to analyze D-pinitol, gas chromatography is a more sensitive technique than with HPLC derivatization, it was decided to develop a GC method.

Based on the determination of D-chiro-lnositol in buckwheat using. HPLC-ELSD was attempted to develop a liquid chromatographic method in parallel with the gas chromatographic method. The first experiments indicated that the repeatability was very low. That is why this method was not further optimized.

Thus, gas chromatography was immediately switched on. It is known to use different columns for the analysis of sugars and sugar derivatives. The table below provides an overview of some of these columns.

Table 3.1

Columns DB-5 and HP-5 (5% phenyl 95% methylplysiloxane) and AT ™ - 5MS / RTX-5MS (5% phenyl polysilphenylene siloxane), of the Alltech type which are moderately polar, and the column AT ™ -1 / RTX-1 (100% methylpolysiloxane) of the Restek type which is a non-polar stationary phase column. Because a decoct is obtained by boiling plant material in water and therefore contains mainly polar substances, it was expected that the analytes would have more retention on the 5% phenyl 95% methylpolysiloxane column than the 100% methylpolysiloxane column, and that this is a better divorce. Therefore, the HP-5 column was chosen over the AT ™ - 1 / RTX-1 column for the decoct analysis. A column with cyanoalkylsilicones as stationary phase is the column AT ™ -264.6% cyanopropylphenyl and 94% methylsiloxane.

An internal standard had to be determined further. Because gas chromatography was used, an internal standard had to be added to correct the variation on the injection. An internal standard must be similar in behavior to the substance to be analyzed. Therefore, some substances with a structure similar to D-pinitol were chosen, namely sugar alcohols such as sorbitol, mannitol, dulcitol, xylitol, and inositol, which are shown below.

However, monosaccharides such as lactose were not chosen because they gave two peaks in the chromatogram due to anomerization. This increases the chance of interference with the internal standard, i.e. the chance that peaks of the steel overlap with those of the internal standard is greater. With disaccharides there is always a risk of degradation, which is difficult to control and is not desirable for a quantitative analysis. High molecular mass sugars or sugar derivatives in comparison with D-pinitol elute late and thus extend the duration of the analysis, so that this compound was not chosen either.

Of the five selected sugar alcohols, dulcitol and inositol are poorly soluble in methanol, but they are soluble in water. These substances are not a first choice because water and also traces of water can cause degradation of the TMS derivatives, which does not benefit a quantitative analysis. Another advantage is that water evaporation takes longer than methanol. In addition, very small amounts, i.h.b. traces, water cannot be seen with the naked eye and a drying agent should be used. The influence on the analysis of this substance must therefore be checked.

Mannitol, sorbitol and xylitol are soluble in methanol, but the retention time of manni, tol and sorbitol overlaps with that of another unknown in the sample as shown in Table 3.2 below. Xylitol elutes at a time where no other substance elutes in the chromatogram and only noise is visible. Also structurally xylitol is a better choice than mannitol or sorbitol because xylitol contains the same number of hydroxyl groups as D-pinitol. This is important because the derivatization reaction continues at the hydroxyl groups. Table 3.2 below contains an overview of the retention times of the possible internal standards.

Table 3.2

With regard to derivatization, sugar derivatives can be derivatized into volatile derivatives by acetylation or silylation. Various reagents are used in the literature, including BSTFA + TMCS in pyridine, HMDS + TFA, HMDS + TMCS in pyridine, STOX + HMDS + TFA, TMSI + pyridine, acetic anhydride + pyridine, and AcO-N-methylimidazole. Among all these options, it was chosen to perform and compare acetylation with acetic anhydride and pyridine and trimethylsilylation with BSTFA + 1% TMCS (+ pyridine).

The acetylation was carried out with acetic anhydride and pyridine in a ratio of 2: 1. The derivatization mixture was added as the internal standard at the weighed amount of solid, D-pinitol, and sorbitol. The correct volume of derivatization mixture had to be found for D-pinitol to be soluble in it. The mixture was either heated in the oven at 60 ° C for 30 minutes or the vial was stored at room temperature overnight. After derivatization, the samples were evaporated to dryness under a stream of nitrogen, after which the derivatives were redissolved in ethyl acetate and analyzed. An AT ™ -264 column was used for this purpose, by analogy with the literature where, before analysis of acetylation derivatives (Ac 2 O-N-methylimidazole), a DB-225 column is used. The temperature of the oven was 200 ° C and was increased at 5 ° C / minute to 220 ° C which was held for 20 minutes.

The peaks had a very large variation in retention time and the repeatability of the peak areas left something to be desired. An explanation could be that the derivatives are relatively not volatile enough, since five hydroxyl groups have to be derivatized.

When trimethyl silylation was used, D-pinitol and sorbitol were weighed and dissolved in 2 ml of methanol. Sorbitol was used for the first derivatization experiments, only later the internal standard was chosen as described above. 100 µl was evaporated to dryness under a stream of nitrogen and 100 µl of BSTFA + 1% TMSC and 40 µl of pyridine were added. The vial was stored in an oven at 70 ° C for 3 hours. The derivatization reagent was then evaporated and the residue redissolved in 300 μΙ hexane: Each level was weighed once but derivatized in duplicate and injected in triplicate. The reaction of the derivatization of a substance with hydroxyl group with BSTFA is shown below:

Table 3.3 below shows an overview of concentration ratio and surface ratio for D-pinitol / internal standard.

Table 3.3

Figure 4 shows a calibration line obtained after derivatizing with BSTFA, which looks linear. At first glance, this method seems linear and the retention time remains the same. From the results of the first experiments it could be deduced that the cjerivatization method is more successful than the acetylation. Work was also continued with this derivatization method.

Thus, with xylitol as the chosen internal standard, the derivatization was further examined. It was also checked whether the reaction time changes slightly on the surfaces.

A methanolic D-pinitol and xylitol solution was prepared at (25 mg / 50 ml) and (10 mg / 50 ml), respectively. In graduated flasks of 10 ml, 2 ml of internal standard and a different amount of D-pinitol were added. Then it was diluted. 500 µl of this solution was evaporated to dryness under a stream of nitrogen. 0.1 ml of derivatization mixture was added to each vial and the vials were placed in an oven at 70 ° C for 3 hours, 6 hours and overnight. Afterwards, the derivatization reagent was evaporated, the residue redissolved in 300 μΙ hexane and injected into the GC in duplicate.

The table 3.4 below gives an overview of the concentration ratio, and surface ratio after three hours of derivatization indicating linearity.

table 3.4

Figure 5 is a graphical representation of the concentration and surface ratios after 3 hours of derivatization.

Table 3.5 below provides an overview of the concentration and surface ratios after 6 hours of derivatization, while Figure 6 is a graphical representation of the concentration and surface ratios after 6 hours of derivatization.

table 3.5

Table 3.6 below provides an overview of the concentration and surface ratios after overnight derivatization, while Figure 7 is a graphical representation of the concentration and surface ratios after overnight derivatization.

table 3.6

Regardless of the derivatization time, there is a large inter-variability for a point with the same concentration as visible in Figures 5 to 7 and in the 3 tables above. No conclusions could be drawn from this regarding the completeness of the derivatization reaction. Another problem was that when samples were left in the oven overnight, there were vials that were empty in the morning and whose derivatizing reagent was probably evaporated. This is also not desirable when measuring quantitatively. Deregulating steel for 6 hours is very difficult in practical terms.

It was then decided to use a heating block to prevent this. Reaction vials were also used, which better conduct the heat. With this method, one hour of derivatization would suffice. Of a solution of D-pinitol and xylitol of 12.5 mg / 100 ml and 10 mg / 50 ml, respectively, 2 ml of internal standard and a different amount of D-pinitol were pipetted in 10 ml volumetric flasks and diluted. 50 μΙ of this solution was evaporated to dryness under a stream of nitrogen. 50 μial of derivatization mixture was added to each vial and the vials were placed in the heating block for one hour at 70 ° C. Afterwards 100 μΙ hexane was added, vortexed and injected into the GC in duplicate. Table 3.7 below shows an overview of the concentration and surface ratios after an hour of derivatization in a heat block while Figure 8 is a graphical representation thereof.

table 3.7

In a subsequent experiment, a higher concentration of xylitol was used and no more hexane was added after the derivatization reaction was continued. This avoided an extra step in the analysis procedure. Table 3.8 below shows an overview of the concentration and surface ratios after an hour of derivatization in a heat block.

table 3.8

When the derivatization reaction proceeds in reaction vials and in a thermal heating block, there are no longer any points of the calibration line that pop out. There is therefore a clear difference with regard to the use of the oven. When both graphs are compared, it appears that the addition of hexane has no major influence on the method. However, it was decided to skip this step in order not to use unnecessary solvent and to make the analysis less labor-intensive.

Now the extraction. To work quantitatively, the extraction of the sample must also be complete. In the following experiment, the best extraction method was sought.

In a first experiment, extraction was carried out by means of an ultrasonic vibrating bath. Approximately 100 mg of steel was weighed for this. 2 ml of internal standard xylitol (18 mg / 50 ml) were added and diluted to 10 ml with methanol. 50 μΙ of this was evaporated to dryness, derivatized and injected into the GC. The table below gives an overview of the surface ratio of D-pinitol / xylitol when extracting 2, 3 or 4 times, and when more volume is used. Table 3.9 below shows an Ü-pinitol / internal standard surface ratio per 100 mg sample after extraction several times on an ultrasonic vibrating bath, while Figure 10 is a graphic representation of this, where 1 stands for 2x extraction, 2 for 2x extraction in 20 ml instead of 10 ml, 3 for 3x extraction and 4 for4x extraction, resp.

Table 3.9

It can thus be concluded from the table above and Figure 10 that the extraction is not yet complete after two or even three times extraction. To know if the extraction is complete after four times. she had to be executed a fifth time. This was a process that was too labor-intensive and further research was carried out into how the substance is best extracted. A larger volume also plays a role: when the volume doubles, the surface ratio also increases.

In a subsequent experiment, the samples were dissolved in 50 ml and 100 ml of methanol, together with the same internal standard as before. The samples were 30 minutes resp. vibrated for one hour on the ultrasonic vibrating bath. Afterwards, a certain amount was evaporated to dryness, derivatized and analyzed. Table 3.10 below shows the data, which gives an overview of the D-pinitol / internal standard surface area per 100 mg sample after extraction several times on one ultrasonic vibrating bath.

Table 3.10

Figure 11 is a graphical representation thereof, in which 1 stands for 50 ml, 30 minutes, 2 for 50 ml, 1 hour, 3 for 100 ml, 30 minutes and 4 for 100 ml, 1 hour, respectively.

When a volume of 50 ml is chosen, the ratio no longer increases when the volume is increased. There is, however, a significant difference between extracting 30 minutes or an hour, as shown in Table 3.10 and Figure 11. However, the extraction is also not complete in this way, since the ratio is even smaller than that after four extractions in 10 ml of methanol.

From previous experiments it appears that especially time vs. extracting the number of times is an important factor. But when a sample is in the vibrating bath, it also heats up, so that the heat factor must also be checked. The table below gives an overview of the surface ratio D-p.initol / internal standard per 100 mg sample after extracting several times on a heated ultrasonic vibrating bath.

Table 3.11

Figure 10 is a graphical representation thereof, in which 1 stands for 1x extracting, 2 for 2x extracting, 3 for 3x extracting and 4 for 4x extracting, resp.

On average, extraction in a heated vibrating bath gives a maximum yield after twice extraction. After extracting one, three or four times, the ratio no longer increases. The ratio after twice heated ultrasonic vibrations is remarkably greater than once an hour extracting without heat, as expected from Table 3.11 and Figure 12. To investigate the influence of heat, another technique, i.e. refluxing, also applied. The table below gives an overview of the surface ratio of D-pinitol / internal standard per 100 mg of steel after extracting several times on a heated ultrasonic vibrating bath.

Table 3.12

The ratios are obtained by means of refluxes are comparable after one, two, three and four refluxes and also when refluxing once for an hour as shown in table 3.12 and figure 13. To get close to this value, vibrations must be performed at least twice in an ultrasonic vibrating bath. The values obtained with reflux are also slightly higher. Whether this is a coincidence cannot be confirmed from these two experiments. However, this shows that refluxing is the most cost-effective and least labor-intensive extraction method as shown in Figure 14.

When working out a final method, the internal standard solution was first looked at, with 10.5 mg of xylitol weighed in a flask on a balance and dissolved in methanol. It was diluted to 100 ml. 25 ml of this solution was pipetted into a 250 ml graduated flask and diluted to 250 ml. The amount of the internal standard gave a ratio of D-pinitol / xylitol in the steel equal to 1.

Next, for the sample preparation, 100 mg of the decoct was weighed in a round bottom flask. Then 50 ml of the internal standard solution was added. This mixture was refluxed for 30 minutes. After cooling, this was transferred to a tube and centrifuged for 5 minutes at 3000 g. The supernatant was transferred to a container. 150 µl of these were pipetted in a reaction vial and evaporated to dryness under a stream of nitrogen. Then 50 μΙ derivatization reagent (BSTFA 1% TMCS - pyridine 2: 1) was added and vortexed. The reaction vials were heated in the heating block at 70 ° C for one hour. After cooling of the vials, the content was transferred to vials that are compatible with the GC auto-injector.

For the analysis, the following temperature gradient was used: during the first two minutes, the temperature was 65 ° C, then the temperature was increased to 300 ° C at 6 ° C / min. 300 ° C was maintained for 15 minutes. A gas with a flow of 1.3 ml / min was used.

Now the method validation. The validation of analytical methods was done according to the ICH guidelines. According to these guidelines the linearity, repeatability and intermediate precision, accuracy, specificity and range must be evaluated for an assay.

When determining the calibration model and range, linearity should be understood to mean that results are obtained that are proportional to the analyte's concentration in the sample. The, range is the interval between the lowest and highest analyte concentration within which it has been demonstrated that the analytical method is accurate, precise and linear.

The response function was determined by injecting 5 standards, with a concentration between 40 and 200% of the theoretical value, in duplicate. To check whether the calibration model is linear, the calibration line is visually inspected and linear regression analysis is performed.

Figure 15 shows that the response curve looks linear and is therefore a straight line.

Table 3.13

Table 3.13 shows the application of regression analysis with a t-test on the slope and 95% confidence interval intersection (0.0). Regression analysis shows that the correlation coefficient. 0.999298 is sufficiently high, i.e.> 0.99. Table 3.13 above shows that there is a significant slope of the line, what. it is also clearly visible in figure 15. When the 95% confidence interval of the intersection is calculated, it is clear that the line does not pass through the point (0,0).

The residues y, - <y,> are plotted against x, or <y,> to check whether the residues are randomly distributed, i.e. homoscedasticity applies. FIG. 16 shows that the residues are evenly distributed and the model is homoscedastic. The residue that deviates the most still has a deviation of less than 5% from the expected value.

An ANOVA lack of fit test was performed to check whether the model is correct. If the average of the two measured values - per concentration - deviated too far from the calibration line with respect to the variance between the two measured values, the F value would be greater than the critical one and the model error was chosen. The calculated F value is 0.7 and therefore less than 4.534. All this data shows that the calibration model is linear.

With regard to precision or repeatability when analyzes are performed on the same sample with a certain method, the method is expected to always give the same results. For this, the precision or repeatability of the method is checked at different levels. The repeatability on the injection was determined by injecting the same sample 6 times. Six samples were analyzed on three different days to check repeatability within a day and intermediate precision.

Precision was also analyzed at various concentration levels, namely the lowest and highest concentration of the range, for example 50% and 200% of the theoretical value.

To analyze repeatability on the injection, the following parameters were calculated from the measurement results: the mean, the standard deviation and the relative standard deviation.

Table 3.14 below shows the levels of D-pinitol for six injections of the same sample with average, standard deviation and relative standard deviation expressed in percent.

It follows from this Table 3.14 that the standard deviation and the relative standard deviation are very small, i.e. the injection is repeatable.

Table 3.14

In addition to repeatability and intermediate precision, in addition to the same calculations as with the precision of the injection, the 95% confidence interval, the intraday and intermediate day variation are also calculated. To this end, a unifactorial variance analysis is performed, namely an ANOVA single factor test. For this, it must first be checked whether the variances of the groups do not differ significantly from each other, otherwise no ANOVA test may be used. Table 3.15 below shows an overview of the levels of D-pinitol on the different days with average, standard deviation, and relative standard deviation expressed in percent.

table 3.15

When this table is considered, it appears that the standard deviations are small and that of day 2 is smaller, approximately half, than that of the other days. The variance coefficients are also rather small. To determine if there is a difference between the results of the different days, an ANOVA test was performed. Before performing this test, it had to be checked whether the variances were the same. The Cochran test is used for this, the formula (1) of which is given below:

(1)

Table 3.16 below shows a summary of the variances for the different days and calculated and critical Cochran value.

, 7

Table 3.16

This table shows that the calculated Cochran value is smaller than the critical value. M.a.w. the variances are considered equal, which means that an ANOVA test can be performed.

Table 3.17 below shows an overview of the variance analysis: squares sum, degrees of freedom, average squares and F values.

Table 3.17

The calculated F value is greater than the critical F value. From this it can be concluded that there is a difference between the results of the different days. To check whether the deviation is still acceptable, the RSD% tUssen is compared with the 2/3 RSD% Horwitz> which gives an estimate of the maximum RSD% that may be made in the same lab. The formula (2) only takes the concentration into account and is the following:

with C the concentration (m / m) (2)

Table 3.18 below shows standard deviations, relative standard deviations expressed in percent, RSDHOrowitz and maximum relative standard deviations.

Table 3.18

This table shows that the RSD% between is smaller than the RSDmax, from which it can be concluded that the method is accurate, despite differences being demonstrated with the ANOVA test. Since the RSD% on the results within the same day are very small, the chance that a significant difference will be found with the ANOVA test increases. Figure 17 is a graphical representation of the individual measurements and averages on different days, showing that the measurements overlap.

Table 3.19

The intermediate precision at different concentration levels has been investigated below. Table 3.19 above shows the levels of D-pinitol in 50 mg, 100 mg and 200 mg of steel with the average, standard deviation and relative standard deviation per series. Thus, this table 3.19 gives an overview of the levels of D-pinitol on the different days with average, standard deviation, and relative standard deviation expressed in percent.

This table shows that the error on the standard deviation with respect to each average is everywhere in the same order of magnitude. To determine whether there is a significant difference between the measurements at different concentration levels, an ANOVA test was also performed here after it was confirmed that the variances are not significantly different.

Table 3.20 below shows a summary of the variances for the different days and calculated a critical Cochran value.

Table 3.20

Here too, the calculated Cochran value is smaller than the critical value from which it can be concluded that no significant difference between the variances of the different groups can be demonstrated.

Table 3.21 below shows an overview of the variance analysis: squares sum, degrees of freedom, average squares and F values.

Table 3.21

It follows from the ANOVA test in table 3.21 above that the calculated F value is greater than the critical F value and that there is therefore a significant difference between the different groups. Graphically the results at 50% and 200% do not overlap with all results at 100%. Nevertheless, the variation between the groups is still acceptable for the method, since the relative standard deviation expressed in percent, the variance coefficient, is smaller than the RSDmax, the. maximum deviation that can be found in a lab.

Table 3.22 below shows standard deviations, relative standard deviations expressed in percent, RSDHrowitz and maximum relative standard deviations.

Table 3.22

When the graph of Figure 16 is considered, it can be concluded that there is no trend with regard to the concentration levels. It can also be concluded from this that sufficient solvent is used and there is no problem with solubility, otherwise the values would increase as the number of mg of sample decreases.

Regarding accuracy, three types of test set-ups are possible to determine whether the value being measured is also the correct value: the test mix method, the standard addition method and the comparison with a generally accepted method. Only the standard addition method can be used here, since the plant material cannot be used as a reconstituted product, since all components are not known, and there is no acceptable method yet, since one had to be developed. The standard addition method means that a known amount of standard solution is added to a quantity of sample. Then it is checked how much of the substance is found according to the following formula:

Table 3.23 below shows the recovery values with mean, standard deviation, relative standard deviation and 95% confidence interval.

Table 3.23) /

Corresponding Figure 19 is a graphical representation of the recovered values after standard addition method, in which 1 = 50% added; 2 = 100% added; 3 = 125% added.

Table 3.24 below shows the recovery values with mean, standard deviation, residual standard deviation and 95% confidence interval.

Table 3.24

As also on the corresponding Figure 20 which represents the recovered values after standard addition method, in which 1 = 50% added; 2 = 100% added; 3 = 125% added, the values where 50% were added are slightly lower than where 100% or 125% were added. To determine whether this is just variation, the experiment had to be repeated again. In general, slightly more than 100% was recovered, which must be settled when analyzes are performed. It should also be noted that the recovery experiment only reveals relative systematic errors and not absolute ones, since the matris is the same before and after adding.

Regarding specificity, the analysis was repeated with mass spectrometric detection to see if only the analyte was measured and not other substances. The analysis was also carried out without an internal standard to verify that no other substance eluted at the same time. Figure 21 shows a partial chromatogram of the analysis without internal standard. No interferents were visible in 20 to 21 minutes.

Figure 22 shows a partial chromatogram of the analysis with internal standard. There are no interferents at the place where xylitol is in the chromatogram.

The mass spectroscopic analysis also confirmed that the method is specific. The corresponding mass spectra are shown in the relevant Fig. 25 et seq. Where Fig. 23 shows a complete chromatogram of the analysis without an internal standard that shows the voltage in mV instead of. minutes.

FIG. 24 shows a complete chromatogram of the analysis of the standards that indicate the voltage in mV i.f.v. minutes, where the Peak at Rt = 20 minutes is Xylitol, while the peak at Rt = 22.45 is D-pinitol. FIG. 25 to 28 tones resp. the specificity via a mass spectrum of standard xylitol; xylitol in steel; D-pinitol steel and standard D-pinitol, which displays the relative abundance in% as a function of the largest signal.

With regard to the in vivo evaluation of the hepatoprotective effect of Desmodium adscendens decoct, D-pinitol has a hepatoprotective effect. One of the ingredients of Desmodium adscendens is D-pinitol. For the in vivo evaluation of the hepatoprotective effect of Desmodium adscendens decoct, previous analyzes show that the decoct of the plant originally contained approximately 0.65% D-pinitol, which is, however, more representative of kidney. In this in vivo experiment, the preventive effect of a decoct of Desmodium adscendens against liver damage induced by galactosamine was investigated in rats. Silymarin is used as a reference agent. Silymarin is a mixture of various flavanolignans from the fruits of the milk thistle, Silybum marianum. The main components are silybine, silychristine and silydianine. In addition, a small amount of isosilybine is found in the milk thistle.

Thus, a number of in vivo experiments in experimental animals were performed with a view to optimizing the dose administered and the treatment schedule and investigating a possible beneficial effect on liver damage due to ethanol but also with acetaminophen.

In the experiment performed the following scheme was used for six groups of test animals: Desmodium decoct 20 mg / kg;

Desmodium decoct 5 mg / kg; D-pinitol 20 mg / kg;

Silymarin 20 mg / kg (positive control); .

y ~

No treatment, only galactosamine intoxication with dose 650 mg / kg (negative control);

No treatment, no intoxication.

The treatment schedule was as follows:

Day 0: pre-treatment with Desmodium decoct: 20 mg / kg or 5 mg / kg Pre-treatment with D-pinitol: 20 mg / kg pre-treatment with Silymarin: 20 mg / kg Day 1: pre-treatment with Desmodium decoct: 20 mg / kg or 5 mg / kg pretreatment with D-pinitol: 20 mg / kg pretreatment with silymarin: 20 mg / kg immediately followed by gelactosamine administration Day 2: all blood collection groups (24 h) Desmodium decoct treatment: 20 mg / kg or 5 mg / kg treatment with D-pinitol: 20 mg / kg treatment with silymarin: 20 mg / kg Day 3: all blood collection groups (48 h).

The following three control groups had to be present in each experiment:

Silymarin 20 mg / kg (positive control);

No treatment, only galactosamine intoxication 650 mg / kg (negative control); ° No treatment, no intoxication.

In the experiment performed, the effect turned out not to be as pronounced after 24 hours, but after 48 hours. For this reason, 24-hour blood collection was not done in follow-up experiments, but only after 48 h, followed by a second blood test after at least another 24 h, and possibly a third blood test at a later time.

Optimization of dose and treatment schedule performed in experiments with D-pinitol. This dose was then calculated to Desmodium decoct for a new experiment.

In order to ascertain the hepatoprotective effect of the preparation to be tested, the selected test animals, namely Sprague Dawley rats, underwent pre-treatment on day 0 and day 1 before the hepatotoxic agent was administered (day 1). On day 2, a one-time follow-up treatment followed. Both the Desmodium decoct, the pure active ingredient D-pinitol, and the positive control silymarin were administered orally by gavage.

This resulted in the aforementioned 6 test groups, viz.

Control no hepatotoxic agent (vehicle), no treatment (vehicle)

Hepatotox: hepatotoxic effect, no treatment (vehicle)

Desmodiuml: hepatotoxic effect, treatment with Desmodium equivalent with 20 mg / kg D-pinitol

Desmodium2 hepatotoxic effect, treatment with Desmodium equivalent with. 5 mg / kg D-pinitol D-pinitol: hepatotoxic effect, treatment with pure D-pinitol 20 mg / kg

Silymarin: hepatotoxic effect, treatment with silymarin 20 mg / kg.

In summary, the treatment schedule was as follows. Pre-treatment was given on day 0 and day 1 with one of the test subjects. After pre-treatment, the relevant groups of test animals were given a hepatotoxic product on day 1: D-galactosamine 650 mg / kg IP 2% in physiol. opl. A treatment dose is also given on day 2. On day 2 and day 3, 1.5 ml of blood was collected from the starting venous. Liver damage and any hepatoprotective effect was evaluated by determining the following parameters in serum: ALT (= GPT), AST (= GOT), ALP.

The results of those tests were as follows for which reference is made to the relevant tables and figures. After administration of the hepatotoxic agent D-galactosamine, a strong increase in the three determined parameters (control vs. hepatotox) could confirm both after 24 hours and after 48 hours of liver damage.

After 24 hours there was no significant decrease for AST (GOT) and ALP for any treatment with respect to the hepatotoxic group, and therefore also not for the positive control silymarin. There was a significant decrease for both Desmodium groups and for silymarin for ALT (GPT).

The results were more pronounced after 48 hours: both AST (GOT) and ALT (GPT) decreased significantly compared to the hepatotoxic group for all treatments: both

Desmodium doses, D-pinitol and Silymarin. Some noticeable findings are that the lowest Desmodium dose (equivalent to 5 mg / kg D-pinitol) already has a very pronounced effect that is comparable to the highest dose (equivalent to 20 mg / kg D-pinitol); that both Desmodium doses are more active than, or at least as active as, silymarin; and that both Desmodium doses are more active than or at least as active as 20 mg / kg of pure D-pinitol.

With regard to the ALP parameter, no effect was yet to be observed after the applied treatment. This may require longer treatment.

Thus, it can be concluded that the hepatoprotective effect of the standardized Desmodium adscendens preparation is clearly demonstrated in the treatment regimen used. Further research into the dose dependence of the effect is recommended, given the good results obtained with the lowest dose of Desmodium decoct. The effect of longer pre-treatment, of a therapeutic treatment that only starts after administration of the hepatotoxic agent, and a combination of both can be further investigated, possibly on a more extensive set of parameters for liver damage.

An experiment consisted of preparing and conducting an experiment with ethanol-induced liver damage - instead of galactosamine -, with the same Desmodium decoct doses as in a previous experiment.

The anti-hepatotoxic activity of a standardized Desmodium adscendens decoct and D-pinitol against chemically induced liver damage in rats was investigated, in particular the protective effect of Desmodium adscendens decoct against ethanol-induced liver damage.

The aim of the experiment is to evaluate the hepatoprotective effects of a decoct of Desmodium adscendens against ethanol-induced liver damage, standardized on its main component D-pinitol.

Materials and methods: 66 male Wistar rats of 200-225g (Charles River, Brussels, Belgium) were randomly divided into 6 groups: the negative control group (CON: no hepatotoxic agent, no treatment: 8 rats), the hepatotoxic group (HEP : induction of hepatotoxicity, no treatment: 20 rats), the Desmodium I group (induction of hepatotoxicity, treatment with Desmodium adscendens decoct, equivalent to 2 mg / kg D-pinitol: 10 rats), the Desmodium II group (induction of hepatotoxicity, treatment with

Desmodium adscendens decoct, equivalent to 10 mg / kg D-pinitol: 10 rats, the D-pinitol group (PIN: induction of hepatotoxicity, treatment with 10 mg / kg D-pinitol: 10 rats), the positive control group (SIL : induction of hepatotoxicity, treatment with 20 mg / kg Silymarin: 8 rats). 2 days before ethanol administration, an appropriate amount of lyophilized decoct, pinitol or Silymarin was suspended in water and administered by gavage (or vehicle for the control group). Hepatotoxicity was induced by daily oral gavage with a 55% ethanol solution (excluding the negative control group). An initial dose of 2 g / kg of ethanol was gradually increased to a dose of 6 g / kg during the first week of the experiment. Ethanol was administered over a period of 7 weeks. Depending on the group, the decoct, pinitol or Silymarin (oral gavage) was specifically treated daily until the end of the experiment. For ethanol administration (baseline), and in weeks 3, 4, 5 and 6, blood samples (1.5 ml) were taken from the lateral tail vein (Multivette 600, Sarstedt). This experiment was approved by the Ethics Committee for Animal Testing at the University of Antwerp (17-01-2011, 2010-37).

A schematic overview of the different treatment groups is given below. Enzyme concentrations of aspartate transaminase (AST, GOT) and alanine transaminase (ALT, GPT) were determined in the serum samples of the test animals by routine laboratory techniques (Senior, 2009). Elevated levels can be considered as an indication of liver cell destruction or a change in membrane permeability.

Statistics: to analyze the difference between CON and HEP for AST and ALT, and between HEP and DES 1, DES 2, PIN and SIL for AST and ALT, a mixed model analysis was performed. The log-rank test was performed for the statistical analysis of the survival data.

Results

Serum AST and ALT values in weeks 4, 5 and 6 of the study are shown in Tables 1-6, and Figures 1-3, 5-7. Average AST and ALT values at different time points (weeks 0, 2-6) are shown in Figures 4 and 8. Tables 7 and 8 and Figure 9 show the survival time in the different animal groups.

Serum AST

Table 2.1

Table 2.1 above shows Serum AST values after 4 weeks of ethanol administration.

Figure 1. shows Serum AST values after 4 weeks of ethanol administration; * p <0.05; ** p <0.01; *** p <0.001 CON versus HEP; + p <0.05 HEP versus DES 1, DES2, PIN, SIL (Mixed model analysis).

Table 2. below shows Serum AST values after 5 weeks of ethanol administration.

Figure 2. shows Serum AST values after 5 weeks of ethanol administration; * p <0.05; ** p <0.01; *** p <0.001 CON versus HEP; + p <0.05 HEP versus DES 1, DES2, PIN, SIL (Mixed model analysis).

Table 3. below shows Serum AST values after 6 weeks of ethanol administration.

!

Figure 3. shows Serum AST values after 6 weeks of ethanol administration; * p <0.05; ** p <0.01; ,, *** p <0.001 CON versus HEP; + p <0.05 HEP versus DES 1, DES2, PIN, SIL (Mixed model analysis).

Figure 4. shows Collected AST values per time point (weeks 0.2.3.4.5.6).

Serum ALT

Table 4. above shows Serum ALT values after 4 weeks of ethanol administration.

Table 5- ,.

Table 5 above shows Serum ALT values after 5 weeks of ethanol administration.

Figure 5. shows Serum ALT values after 4 weeks of ethanol administration; * p <0.05; ** p <0.01; *** p <0.001 CON versus HEP; + p <0.05 HEP versus DES1, DES2, PIN, SIL (Mixed model analysis).

Figure 6. shows Serum ALT values after 5 weeks of ethanol administration; * p <0.05; ** p <0.01; *** p <0.001 CON versus HEP; tp <0.05 HEP versus DES 1, DES2, PIN, SIL (Mixed model analysis).

Table 6. below shows Serum ALT values after 6 weeks of ethanol administration.

Figure 7. shows Serum ALT values after 6 weeks of ethanol administration; * p <0.05; ** p <0.01; *** p <0.001 CON versus HEP; + p <0.05 HEP versus DES 1, DES2, PIN, SIL (Mixed model analysis).

Figure 8. shows Average ALT values per time point (weeks 0.2.3.4.5.6).

Mortality

Table 7. shows Survival of the test animals in the test groups over a period of 6 weeks.

Figure 9. shows survival of the test animals in the test groups over a period of 6 weeks. Table 8. shows Log-rank test for determining difference in survival of the test animals over a period of 6 weeks.

In this experiment, where the preventive effect of Desmodium adscendens decoct against ethanol-induced liver damage was evaluated, the hepatotoxic group (HEP) showed significantly increased serum levels of AST and ALT after 4 weeks of daily ethanol administration (Fig. 1-3,5-) 7). Treatment with Desmodium adscendens (2 mg / kg and 10 mg / kg), pinitol (10 mg / kg) or Silymarin (20 mg / kg) was started 2 days before ethanol administration and continued daily until the end of the experiment, 6 weeks later on. As shown in Figures 1-3 and 5-7, no significant decrease was observed for serum AST (Fig. 1, 2, 3 after treatment of 4, 5 and 6 weeks, respectively) and ALT levels (Fig. 5, 6 and 7, after treatment of 4, 5 and 6 weeks, respectively).

Evaluation of the mortality of the animals in the different treatment groups (Fig. 9; Table 7), showed a large failure in the untreated hepatotoxic group (HEP), while survival in DES 2 was better. In addition, after 6 weeks of ethanol administration, 60% of the rats receiving Desmodium adscendens decoct survived, while survival in the pinitol and Silymarin treated groups was 40% and 22%, respectively. Statistical analysis of survival data (Table 8) showed a statistically significant difference in survival between control and hepatotoxic group (p <0.01), and a trend for significance (p = 0.06) between the Desmodium adscendens group DES 2 (eq 10 mg / kg pinitol) and the untreated hepatotoxic group. No statistical difference of the DES 1 group (eq. At 2 mg / kg pinitol), pinitol group (10 mg / kg) or silymarin group (20 mg / kg) with the hepatotoxic group was observed.

. Since a period of at least 4 weeks was found to cause significant ethanol-induced hepatotoxic effects in rats and a major failure of untreated hepatotoxic animals was observed, it was not possible to investigate the hepatocurative effects of Desmodium adscendens decoct in this experiment .

A further experiment consisted of preparing and conducting an experiment with acetaminophen-induced liver damage instead of galactosamine.

The aim of the experiment was to evaluate the hepato-curative effects of a decoction of Desmodium adscendens, standardized on its main component D-pinitol, against acetaminophen (paracetamol) -induced liver damage.

The materials and methods used for this purpose existed in 40 male 200-225g Wistar rats randomly divided into 5 groups: the negative control group (CON: no hepatotoxic agent, no treatment: 8 rats); the hepatotoxic group (HEP: induction of hepatotoxicity, no treatment: 8 rats); the Desmodium I group (induction of hepatotoxicity, treatment with Desmodium adscendens decoct, equivalent to 2 mg / kg D-pinitol: 8 rats); the Desmodium ll group (induction of hepatotoxicity, treatment with Desmodium adscendens decoct, equivalent to 10 mg / kg D-pinitol: 8 rats); the positive control group (SIL: induction of hepatotoxicity, treatment with 20 mg / kg of silymarin: 8 rats). Acute hepatotoxicity was induced by oral gavage with 2 g / kg acetaminophen (except the control group). 24 h after hepatotoxicity induction, treatment was initiated with the appropriate amount (see above) of lyophilized decoction of Desmodium adscendens or silymarin, suspended in water and administered by gavage (or control vehicle and hepatotoxic groups). Blood samples were taken from the lateral start vein (1.5 ml, Multivette 600, Sarstedt) 24h, 48h and 72h after acetaminophen administration. This experiment was approved by the Ethics Committee for Animal Testing at the University of Antwerp (17-01-2011, 2010-37).

A schematic overview of the different treatment groups is given below: CON: no acetaminophen, no treatment HEP: 2g / kg acetaminophen, no treatment DES 1: 2g / kg acetaminophen, Desmodium adscendens treatment equivalent to 2 mg / kg D-pinitol DES 2: 2g / kg acetaminophen, Desmodium adscendens treatment equivalent to 10 mg / kg D-pinitol SIL: 2g / kg acetaminophen, silymarin treatment (20 mg / kg).

Enzyme levels of aspartate transaminase (AST, GOT) and alanine transaminase (ALT, GPT) were determined in serum samples from laboratory animals with routine laboratory techniques (Senior, 2009). Elevated levels can be considered as an indication of liver cell destruction or a change in membrane permeability.

Statistically, to analyze the difference between CON and HEP for AST and ALT, and between HEP and DES 1, DES 2, and SIL for AST and ALT, a One-way ANOVA was performed, followed by Dunnett post-hoc test ( SPSS statistics program).

The results for Serum AST and ALT values at 48h and 72h after acetaminophen administration are shown in Tables 1-4, and Figures 1-3.

Table 1

Table 2

Figure, shows Serum AST values 48h after acetaminophen administration; * p <0.05; ** p <0.01; *** p <0.001 CON versus HEP (Statistics: One-Way Anova, Dunnett post-hoc versus HEP).

Serum ALT

Tables 3 & 4 show Serum ALT values 48 and 72h after acetaminophen administration.

Figures show Serum ALT values 48h and 72h after acetaminophen administration; * p <0.05; ** p <0.01; *** p <0.001 CON versus HEP (Statistics: One-Way Anova, Dunnett post-hoc versus HEP).

In this experiment, which evaluated the curative effect of Desmodium adscendens décoct against acetaminophen-induced liver damage, the hepatotoxic group (HEP) showed significantly increased levels of serum AST and ALT at 24h and 48h after acetaminofen administration (p <0.001), and also at 72h after acetaminophen administration for ALT (p <0.05). An oral treatment of Desmodium adscendens (2 mg / kg and 10 mg / kg) or Silymarin (20 mg / kg, positive control group) was given 24h and 48h after acetaminophen administration. As shown in Figs. 1-3, no significant decrease (p> 0.05) could be observed for serum AST and ALT levels at blood sampling points 48h and 72h for any treatment. The results of this experiment in a rat model of acute liver damage, induced by acetaminophen, show that Desmodium adscendens has no hepatocurative effect when administered at a daily dose of up to 10 mg / kg.

Finally, a so-called AMES test was conducted on steel UA: Desmodium extract. The extract was tested according to the OECD guideline with 5 Salmonella typhimurium strains (TA98, TA100, TA102, TA1535 and TA1537) in the presence and absence of a metabolising S9 fraction. New strains, resp. also S9 and other supplies such as NADPH, petri plates, etc.

In accordance with the guidelines, a maximum test concentration of 5 mg / plate was used, after which dilutions were carried out with 6 doses. A negative (solvent) control was used with 4 petri plates, as well as a positive control with 3 petri plates. Positive controls are part of the recommended list of controls at the recommended test concentration. There is a list of positive controls that we used, and their concentration.

Thus, three petri plates were made for each concentration and 4 for the negative control. The results represented the average of these, in particular average number of revertants ± SD. The test was successful when the negative and positive concentrations were within the confused range and there were no signs of toxicity. This was checked by checking the background layer of bacteria in which the absence of a background layer indicates toxicity. No signs of toxicity were found and all strains and controls gave the expected response to TA102. A test substance is positively "mutagenic" when a doubling of the number of revertants is observed relative to the negative (solvent) control and when a dose-effect relationship is observed.

A synthesis of the results is shown below.

The above data indicate that the sample is not mutagenic in strains TA98, TA100 and TA1535, both in the presence and absence of S9. For TA1537, in the presence of S9, at the highest concentration t.w. 5 mg / plate, just doubling the mutation frequency found relative to the negative control, but no pronounced dose-effect relationship. The test was repeated to be sure. No dose-effect relationship was found and no doubling compared to the control, which leads to the conclusion that no mutagenic effect was found in strain TA1537 either.

It could be determined experimentally that there is virtually no mutagenic effect. The Desmodium extract is not mutagenic in the Ames test, neither in the absence nor in the presence of a metabolising S9 fraction. An increase in the number of spontaneous revertants can be observed at the highest dose, but it was nevertheless insufficient to be able to speak of mutagenicity. Repeat experiments (at least 1 per strain) confirm the absence of mutagenicity.

Claims (39)

A method for manufacturing a pinitol-quantified plant extract, wherein a plant is selected from the Desmodium family Desmodium adscendens, wherein a fraction of plant parts Desmodium is isolated, wherein a plant extract is derived from said fraction thereof, characterized in that a characterized extract is derived from Desmodium adscendens from which a preparation of said plant extract is quantified for pinitol. Method according to claim 1, characterized in that a validated analysis method is used for said quantification of Desmodium adscendens on pinitol, i.h.b. D-pinitol, in which a specifically quantified Desmodium adscendens preparation is produced. Method according to the preceding claim, characterized in that a biologically controlled isolation is carried out in the aforementioned analysis method which is developed with obtaining a preparation quantitated for D-pinitol, the preparation product being enriched each time and numerous fractions being isolated, and wherein decoct is obtained which contains an amount of D-pinitol. Method according to one of the preceding claims, characterized in that the constituents of said preparation are standardized with determination of the reproducibility of the production process. Method according to one of claims 2 to 4, characterized in that the main components of the components in Desmodium adscendens and the total amount of D-pinitol are determined by means of an analytical method adapted for this purpose, i.h.b. wherein the extraction process, the products obtained after extraction and the conditions are evaluated and optimized. Method according to one of the preceding claims, characterized in that said plant extract is derived from a fraction consisting of aboveground parts of Desmodium adscendens, either including leaves, branches, stems and / or flowers or fruits thereof. Method according to one of the preceding claims, characterized in that said plant extract is derived from a fraction consisting of aboveground parts of Desmodium adscendens, either including flowers or fruits. A method according to any one of the preceding claims, characterized in that said plant extract is derived from a fraction consisting of underground parts of Desmodium adscendens or including their roots. Method according to the preceding claim, characterized in that said plant parts are dried beforehand. Method according to one of the preceding claims, characterized in that said plant parts are subsequently made finer or fragmented. Method according to the preceding claim, characterized in that said plant parts are pulverized in powder form. Method according to the preceding claim, characterized in that an extract of said plant parts of Desmodium adscendens is prepared by extraction with, inter alia, methanol and / or ethanol. A method according to the preceding claim, characterized in that an extract of said plant parts of Desmodium adscendens is prepared by extraction with water. Method according to one of the two preceding claims, characterized in that an extract of said plant parts of Desmodium adscendens is prepared by extraction with less polar or non-polar solvents such as but not exclusively n hexane or combinations thereof. A method according to any one of the preceding claims, characterized in that an aqueous decoction of said plant parts of Desmodium adscendens is then hot prepared to a decoct by boiling a certain amount of dried, possibly pulverized plant parts in a certain amount of water, i.h.b. distilled water for a certain period of time. Method according to the preceding claim, characterized in that said aqueous decoction of said plant parts of Desmodium adscendens is subsequently cooled, after which the portions obtained are combined and filtered, after which the resulting filtrate is concentrated, i.h.b. under vacuum, and then lyophilized. Method according to claim 15, characterized in that said aqueous decoction of said plant parts of Desmodium adscendens is subsequently cooled, after which the portions obtained are combined and filtered, after which the resulting filtrate is concentrated, i.h.b. under vacuum, and then spray dried. The method according to any one of claims 1 to 14, characterized in that an aqueous product of said plant parts of Desmodium adscendens is then cold-prepared into an extract, i.h.b. by incorporating a certain amount of dried, possibly pulverized, plant parts into a certain amount of water, i.h.b. distilled water for a certain period of time. Method according to one of claims 9 to 18, characterized in that starting from 1 kg of dried plant parts of 60 to 70 g, i.h.b. 65 g of extract is obtained, resp. in a ratio of 10-20: 1, in particular 14-16: 1. Method according to one of claims 7 to 19, characterized in that subsequently a certain amount of said extract is subjected to column chromatography, with methanol elution, whereby certain fractions are collected and analyzed by thin layer chromatography, and MeOH / H 2 O: 5: 1 as a mobile phase, and where their chromatographic pattern is determined. A method according to the preceding claim, characterized in that about 20 g, said extract is subjected to column chromatography on Sephadex LH-20 with methanol elution, wherein about 100 ml are collected and analyzed with thin layer chromatography-silica gel with a layer thickness of approx. 0.25 cm, and MeOH / H2 O: 5: 1 as the mobile phase, and their chromatographic pattern being determined based on these parameters. Method according to one of the two preceding claims, characterized in that said fractions are combined in a number, i.h.b. 11, subfractions according to their chromatographic pattern. Method according to the preceding claim, characterized in that said subfractions 5-11, i.h.b. 200 mg, showing a stain with a green color, after spraying with anisaldehyde 1% H 2 SO 4 in MeOH, and heating to about 120 ° C for 10 minutes, are combined, this fraction being subjected to a further column chromatography eluted with MeOH, where again certain fractions, in particular of 100 ml, collected and analyzed. The method according to the preceding claim, characterized in that said subfractions 4-5, i.h.b. 120 mg of this column are combined, whereby they are subjected to a further column chromatography under the same conditions, after which a white product is obtained. Method according to any of claims 3 to 24, characterized in that said extract is isolated and the isolated product is identified as the methylated cyclitol 3-O-methyl-chiro-inositol, namely D-pinitol. Process according to the preceding claim, characterized in that sugar alcohols are chosen as internal standard, in particular xylitol, in particular in gas chromatography. A method according to the preceding claim, characterized in that other sugar alcohols such as sorbitol, mannitol, and, if appropriate, dulcitol and inositol, instead of the aforementioned xylitol, are also chosen to a lesser extent. Method according to one of the preceding claims, characterized in that L-pinitol is produced from said extract from which it is separated. A method according to any one of the preceding claims, characterized in that a plant extract of Desmodium adscendens of tropical origin is used. Method according to one of claims 1 to 29, characterized in that a standardized extract is derived from the Desmodium adscendens plant, quantified for D-pinitol in its action against hepatitis, i.h.b. preventively, with isolation of D-pinitol from Desmodium adscendens, where D-pinitol forms an active ingredient. A method according to any one of claims 1 to 29, characterized in that a standardized extract is derived from the plant Desmodium adscendens, quantified for D-pinitol in its curative action against hepatitis, with separation of D-pinitol from Desmodium adscendens, wherein D -pinitol is an active ingredient. A plant extract obtained according to a method as defined in any one of the preceding claims for use in a method of protecting the liver in a mammal, to prevent liver disease in a mammal. A plant extract obtained according to a method as defined in any one of claims 1 to 31 for treatment of a liver disease in a mammal. Extract from a plant according to one of the two preceding claims, characterized in that it is standardized to be derived from plant Desmodium adscendens, quantified on the molecule D-pinitol active against hepatitis, wherein D-pinitol is isolated from Desmodium adscendens where D-pinitol is a active ingredient. Extract from a plant according to any of claims 32 to 34, characterized in that the plant extract comprises between 0.1 and 10%, preferably from 4 to 5% pinitol, i.h.b. D-pinitol. A plant extract according to any one of claims 32 to 35, characterized in that the D-pinitol-containing plant extract is given to a mammal in the form of a composition containing it, said composition being selected from the group consisting of a pharmaceutical composition, a food composition and / or a beverage composition. An extract from a plant Desmodium adscendens according to any of claims 32 to 36, for use in a method of prevention of a liver disease in a mammal, which comprises the step of administering an effective amount of said extract of Desmodium adscendens thereto . An extract from Desmodium adscendens according to any one of claims 32 to 37, for use in a method of treatment of a liver disease in a mammal, which comprises the step of administering an effective amount of said extract of Desmodium adscendens thereto. Use of a plant extract as claimed in any one of claims 32 to 38, characterized in that it is used as an antioxidant intended for specific target groups, i.h.b. the intended target group being human, more i.h.b. by alcoholics and / or diabetes type 2 diabetes patients.